Because of their non-invasiveness and non-ionization nature, therapeutic ultrasound and focused shock waves have been used extensively in clinic for the treatment of cancers, targeted drug delivery (such as opening of blood-brain-barriers), disintegration of concretions, fractionation of soft tissues, and healing of non-union fractures, a well as pain therapy. Although in all these applications cavitation has often been implicated as a key mechanism whereby the desirable therapeutic effects and, in some cases, the undesirable adverse effects are produced, the dynamic processes of bubble(s)-tissue interaction and, in particular, bubble(s)-cell interaction, are largely unknown. This is primarily due to the lack of viable experimental systems that can be used to investigate such dynamic interactions with sufficient spatial and temporal resolutions. To overcome this primary challenge, we propose to develop a new experimental system and associated technologies to investigate the bioeffects produced by cavitation bubbles at the single cell level. This project will be carried out by a multidisciplinary research team with recognized expertise in therapeutic ultrasound and cavitation (Zhong Lab) and cell mechanics (Guilak Lab). Two specific aims are proposed: 1) development of a microfluidic based system that allows for precise control of the location and orientation of individual cells grown in each channel, as well as their spatial alignment with laser- and ultrasound-generated cavitation bubbles, 2) investigation of the bioeffects (cell lysis, membrane poration, Ca2+ influx, cytoskeleton re-arrangement, injury repair, viability, and proliferation) and mechanical deformation of the cell produced by the bubble(s)-cell interaction using representative cell lines (i.e., HeLa cells, MDCK epithelial cells, and chondrocytes) relevant to therapeutic ultrasound and shock wave therapy. With a better understanding of the bubble(s)-cell interaction and the resultant mechanical and biological consequences elicited in different cell types, we hope to gain insights that may be used in the future to improve the design of therapeutic ultrasound and shock wave devices, as well as treatment protocols for safe and more effective medical applications. Furthermore, the new knowledge acquired from this project may be used to develop novel ultrasonic techniques that can be applied to stimulate stem cells under precise mechanical loading conditions to impact their differentiation, growth and phenotypic expression with tissue engineering and regenerative medicine applications. The goal of this R03 application is to demonstrate the feasibility in constructing such a novel experimental system and developing associated technologies for assessing the bioeffects produced by cavitation bubbles at the single-cell level, while the comprehensive utilization of this novel system will be exploited in a follow-up RO1 application.